Methods for treating cancers and pathogen infections using antigen-presenting cells loaded with RNA

ABSTRACT

Disclosed are cells and methods for treating or preventing tumor formation or infections with pathogens in a patient. The cells of the invention are antigen-presenting cells (e.g., dendritic cells or macrophage) that have been loaded with RNA derived from tumors or pathogens. By administering the RNA-loaded antigen-presenting cells to a patient, tumor formation or pathogen infections can be treated or prevented. Alternatively, the RNA-loaded cells can be used as stimulator cells in the ex vivo expansion of CTL. Such CTL can then be used in a variation of conventional adoptive immunotherapy techniques.

This application is a continuation of application Ser. No. 12/585,028,filed Sep. 1, 2009, now U.S. Pat. No. 8,263,066, which is a divisionalof application Ser. No. 11/250,546, filed Oct. 17, 2005, now U.S. Pat.No. 7,601,343, which is a continuation of application Ser. No.09/171,916, filed Feb. 16, 1999, now U.S. Pat. No. 7,105,157, which is a371 of International Application No. PCT/US97/07317, filed Apr. 30,1997, which designated the U.S. and is a continuation-in-part of U.S.application Ser. No. 08/640,444 filed Apr. 30, 1996, now U.S. Pat. No.5,853,719. The entire contents of which are hereby incorporated byreference in this application.

BACKGROUND OF THE INVENTION

This invention relates to methods for treating or preventing tumorformation or pathogen infection in a patient.

Previously-described methods for treating cancers include the use ofchemotherapeutics, radiation therapy, and selective surgery. Theidentification of a few tumor antigens has led to the development ofcell-based therapies. These methods rely on first identifying a tumorantigen (i.e., a polypeptide that is expressed preferentially in tumorcells, relative to non-tumor cells). Several human tumor antigens havebeen isolated from melanoma patients, and identified and characterized(Boon and van der Bruggen, 1996, J. Exp. Med. 183: 725-729). Thesepolypeptide antigens can be loaded onto antigen-presenting cells, andthen be administered to patients in a method of immunotherapy (i.e., asa vaccine). Alternatively, the polypeptide-loaded antigen-presentingcells can be used to stimulate CTL proliferation ex vivo. The stimulatedCTL are then administered to the patient in a method of adoptiveimmunotherapy.

A variety of methods have been described for treating infections withintracellular pathogens such as viruses and bacteria. For example,antibiotics are commonly used to treat bacterial infections.Preparations of killed pathogens can also serve as vaccines. Inaddition, CTL-based therapies have been described for treating suchinfections.

SUMMARY OF THE INVENTION

It has now been discovered that tumor formation in a patient can betreated or prevented by administering to the patient anantigen-presenting cell(s) that is loaded with antigen encoded in RNAderived from a tumor. For convenience, an RNA-enriched tumor preparationcan be used in lieu of purified RNA. The invention thus circumvents theneed purify RNA or isolate and identify a tumor antigen. Using similarmethods and pathogen-derived RNA, pathogen infection in a patient can betreated or prevented. The RNA-loaded antigen-presenting cells can beused to stimulate CTL proliferation ex vivo or in vivo. The ex vivoexpanded CTL can be administered to a patient in a method of adoptiveimmunotherapy.

Accordingly, the invention features a method for producing an RNA-loadedantigen-presenting cell (APC); the method involves introducing into anAPC in vitro (i) tumor-derived RNA that includes tumor-specific RNAwhich encodes a cell-surface tumor antigenic epitope which induces Tcell proliferation or (ii) pathogen-derived RNA that includespathogen-specific RNA which encodes a pathogen antigenic epitope thatinduces T cell proliferation. Upon introducing RNA into an APC (i.e.,“loading” the APC with RNA), the RNA is translated within the APC, andthe resulting protein is processed by the MHC class I or class IIprocessing and presentation pathways. Presentation of RNA-encodedpeptides begins the chain of events in which the immune system mounts aresponse to the presented peptides.

Preferably, the APC is a professional APC such as a dendritic cell or amacrophage. Alternatively, any APC can be used. For example, endothelialcells and artificially generated APC can be used. The RNA that is loadedonto the APC can be provided to the APC as purified RNA, or as afractionated preparation of a tumor or pathogen. The RNA can includepoly A⁺ RNA, which can be isolated by using conventional methods (e.g.,use of poly dT chromatography). Both cytoplasmic and nuclear RNA areuseful in the invention. Also useful in the invention is RNA encodingdefined tumor or pathogen antigens or epitopes, and RNA “minigenes”(i.e., RNA sequences encoding defined epitopes). If desired,tumor-specific or pathogen-specific RNA can be used; such RNA can beprepared using art-known techniques such as subtractive hybridizationagainst RNA from non-tumor cells or against related, but non-pathogenic,bacteria or viruses.

The RNA that is loaded onto APC can be isolated from a cell, or it canbe produced by employing conventional molecular biology techniques. Forexample, RNA can be extracted from tumor cells, reverse transcribed intocDNA, which can be amplified by PCR, and the cDNA then is transcribedinto RNA to be used in the invention. If desired, the cDNA can be clonedinto a plasmid before it is used as a template for RNA synthesis. RNAthat is synthesized in vitro can, of course, be synthesized partially orentirely with ribonucleotide analogues or derivatives. Such analoguesand derivatives are well known in the art and can be used, for example,to produce nuclease-resistant RNAs. The use of RNA amplificationtechniques allows one to obtain large amounts of the RNA antigen from asmall number of cells.

Included within the invention are methods in which the RNA is isolatedfrom a frozen or fixed tissue. Tumor specimens commonly are isolatedfrom cancer patients and then stored, for example, as cryostat orformalin fixed, paraffin-embedded tissue sections. Because cancerpatients often have few tumor cells, the isolation of RNA from fixedtissues is particularly advantageous in producing the APCs of theinvention because the method can utilize a small tissue sample.Microdissection techniques can be used to separate tumor cells fromnormal cells. RNA can then be isolated from the tumor cells andamplified in vitro (e.g., by PCR or reverse transcription PCR (RT-PCR)).The resulting, amplified RNA then can be used to produce the RNA-loadedAPCs described herein.

If desired, RNA encoding an immunomodulator can also be introduced intothe APC loaded with tumor-derived or pathogen-derived RNA. In thisembodiment, the RNA-encoded immunomodulator is expressed in the APC andenhances the therapeutic effect (e.g., as a vaccines) of the RNA-loadedAPCs. Preferably, the immunomodulator is a cytokine or costimulatoryfactor (e.g., an interleukin, such as IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, or IL-15, or GM-CSF).

To introduce RNA into an APC, the APC may be contacted with the tumor-or pathogen-derived RNA in the presence of a cationic lipid, such asDOTAP or 1:1 (w/w) DOTMA:DOPE (i.e., LIPOFECTIN). Alternatively, “naked”RNA can be introduced into the cells. Other art-known transfectionmethods also can be used to introduce the RNA into the APC.

In a variation of the above methods, the RNA that is introduced into theAPC can be engineered such that it encodes a cell trafficking signalsequence in addition to a tumor antigen or pathogen antigen. Such anengineered RNA can be thought of as containing two RNA sequences thatare covalently linked and which direct expression of a chimericpolypeptide. One RNA sequence encodes the tumor or pathogen antigen,while the other RNA sequence encodes the cell trafficking sequence, thusforming a chimeric polypeptide. The chimeric polypeptides that containan antigen linked to a trafficking sequence are channeled into the MHCclass II antigen presentation pathway. Examples of suitable traffickingsequences are provided below.

Because practicing the invention does not require identifying an antigenof the tumor cell or pathogen, RNA derived from essentially any type oftumor or pathogen is useful. For example, the invention is applicable,but not limited, to the development of therapeutics for treatingmelanomas, bladder cancers, breast cancers, pancreatic cancers, prostatecancers, colon cancers, and ovarian cancers. In addition, the inventioncan treat or prevent infections with pathogens such as Salmonella,Shigella, Enterobacter, human immunodeficiency virus, Herpes virus,influenza virus, poliomyelitis virus, measles virus, mumps virus, orrubella virus.

The antigen-presenting cells produced in accordance with the inventioncan be used to induce CTL responses in vivo and ex vivo. Thus, theinvention includes methods for treating or preventing tumor formation ina patient by administering to the patient a therapeutically effectiveamount of APC loaded with tumor-derived RNA. The tumor-derived RNA canbe derived from the patient, e.g., as an RNA-enriched tumor preparation.Alternatively, the tumor-derived RNA used in such a treatment regimencan be derived from another patient afflicted with the same, or asimilar, type of cancer. Likewise, APC loaded with pathogen-derived RNAcan be used to treat or prevent a pathogen infection in a patient.

Included within the invention are methods for producing a cytotoxic Tlymphocyte. Such a CTL can be produced by contacting a T lymphocyte invitro with an antigen-presenting cell that is loaded with tumor-derivedor pathogen-derived RNA, and maintaining the T lymphocyte underconditions conducive to CTL proliferation, thereby producing a CTL. Theresulting CTL show remarkable specificity for the pathogen or the cellsof the tumor from which the loaded RNA is derived. Such CTL can beadministered to a patient in a variation of conventional adoptiveimmunotherapy methods. If desired, one can assay for sensitization(i.e., activation) of the CTL. To this end, any of the art-known assays,such as cytotoxicity assays, can be used. For example, the cytotoxicityassay can include detecting killing of an RNA-loaded cell(s) (e.g.,fibroblasts or dendritic cells produced as described herein (i.e., anRNA-loaded cell) that presents on its surface a tumor antigenic epitopeor pathogen antigenic epitope encoded by the RNA). Another suitablemethod for detecting CTL sensitization is by detecting an increase incytokine secretion, relative to the level of cytokine secretion (e.g.,TNF-α or γ-interferon) obtained prior to contacting the CTL (e.g., in anin situ immunoassay, such as an ELISPOT assay).

In a variation of the above methods, the invention provides a method forgenerating a tumor-specific (or pathogen-specific) CTL response. Becausemost cancer patients naturally display a non-detectable or poortumor-specific CTL response, this method is particularly useful since itprovides a method for producing a CTL response using antigens obtainedfrom any patient. Once a CTL response is generated, one can useconventional methods to identify the antigens that induce the CTLresponse. For example, antigens in tumor extracts can be fractionated(e.g., by HPLC), and the fractions can be assayed to determine whichfraction contains an antigen that is recognized by the tumor-specificCTL produced in accordance with the invention. The method of theinvention thus serves as a platform for antigen discovery. The methodentails:

a) introducing into an antigen-presenting cell in vitro polyA⁺ RNA thatincludes at least 80% (preferably at least 90% or 100%) of the polyA⁺RNA species, thereby producing an RNA-loaded APC; and

b) contacting a T lymphocyte with the RNA-loaded APC, thereby producinga tumor-specific or pathogen-specific CTL response. If desired,induction of such a CTL response can be detected by detectingsensitization of the contacted T lymphocyte using the methods describedherein. In practicing this method, unfractionated, total tumor-derived(or pathogen-derived) RNA typically will be used to produce theRNA-loaded APC, since total RNA is certain to contain an RNA speciesthat encodes the tumor antigen (or pathogen antigen).

The invention also includes methods for treating or preventing tumorformation in a patient by administering to the patient a therapeuticallyeffective amount of APC loaded with tumor-derived RNA. Similarly, theinvention provides methods for treating pathogen infection in a patientby administering to the patient a therapeutically effective amount ofAPC loaded with pathogen-derived RNA. The T lymphocytes that are used inthese various therapeutic methods can be derived from the patient to betreated, or haplotype-matched CTL from a donor can be used. Similarly,the RNA used in these methods can be derived from the patient to betreated, or RNA from a donor can be used.

By “RNA-loaded” or “RNA-pulsed” antigen-presenting cell is meant an APC(e.g., a macrophage or dendritic cell) that was incubated or transfectedwith RNA, e.g., RNA derived from a tumor or pathogen. Such RNA can beloaded onto the APC by using conventional nucleic acid transfectionmethods, such as lipid-mediated transfection, electroporation, andcalcium phosphate transfection. For example, RNA can be introduced intoAPC by incubating the APC with the RNA (or extract) for 1 to 24 hours(e.g., 2 hours) at 37° C., preferably in the presence of a cationiclipid.

By “tumor-derived” RNA is meant a sample of RNA that has its origin in atumor cell, and which includes RNA corresponding to a tumor antigen(s).Included is RNA that encodes all or a portion of a previously identifiedtumor antigen. Similarly “pathogen-derived” RNA is a sample of RNA thathas its origin in an pathogen (e.g., a bacterium or virus, includingintracellular pathogens). Such RNA can be “in vitro transcribed,” e.g.,reverse transcribed to produce cDNA that can be amplified by PCR andsubsequently be transcribed in vitro, with or without cloning the cDNA.Also included is RNA that is provided as a fractionated preparation oftumor cell or pathogen. Because even unfractionated RNA preparation(e.g., total RNA or total poly A+ RNA) can be used, it is not necessarythat a tumor or pathogen antigen be identified. In one embodiment, thepreparation is fractionated with respect to a non-RNA component(s) ofthe cell in order to decrease the concentration of a non-RNA component,such as protein, lipid, and/or DNA and enrich the preparation for RNA.If desired, the preparation can be further fractionated with respect tothe RNA (e.g., by subtractive hybridization) such that “tumor-specific”or “pathogen-specific” RNA is produced.

By “tumor-specific” RNA is meant an RNA sample that, relative tounfractionated tumor-derived RNA, has a high content of RNA that ispreferentially present in a tumor cell compared with a non-tumor cell.For example, tumor-specific RNA includes RNA that is present in a tumorcell, but not present in a non-tumor cell. Also encompassed in thisdefinition is an RNA sample that includes RNA that is present both intumor and non-tumor cells, but is present at a higher level in tumorcells than in non-tumor cells. Also included within this definition isRNA that encodes a previously identified tumor antigen and which isproduced in vitro, e.g., from a plasmid or by PCR. Alternatively,tumor-specific RNA can be prepared by fractionating an RNA sample suchthat the percentage of RNA corresponding to a tumor antigen isincreased, relative to unfractionated tumor-derived RNA. For example,tumor-specific RNA can be prepared by fractionating tumor-derived RNAusing conventional subtractive hybridization techniques against RNA fromnon-tumor cells. Likewise, “pathogen-specific” RNA refers to an RNAsample that, relative to unfractionated pathogen-derived RNA, has a highcontent of RNA that is preferentially present in the pathogen comparedwith a non-pathogenic strain of bacteria or virus.

By “trafficking sequence” is meant an amino acid sequence (or an RNAencoding an amino acid sequence) that functions to control intracellulartrafficking (e.g., directed movement from organelle to organelle or tothe cell surface) of a polypeptide to which it is attached.

The invention offers several advantages. Vaccinations performed inaccordance with the invention circumvent the need to identify specifictumor rejection antigens or pathogen antigens, because the correctantigen(s) is automatically selected from the tumor- or pathogen-derivedRNA when unfractionated RNA is used. If desired, the risk of generatingan autoimmune response can be diminished by using tumor-specific RNA. Inaddition, vaccination with cells loaded with unfractionatedtumor-derived RNA likely elicits immune responses to several tumorantigens, reducing the likelihood of “escape mutants.” The inventionalso extends the use of active immunotherapy to treating cancers forwhich specific tumor antigens have not yet been identified, which is thevast majority of cancers. Furthermore, the RNA to be introduced intoAPCs can be derived from fixed tissue samples. Fixed samples of tumortissues are routinely prepared in the course of diagnosing cancer; thus,the use of RNA from such samples does not require subjecting a patientto an additional invasive procedure. Because most cancer patients havelow tumor burdens, the methods of the invention that involve isolationand amplification of RNA from fixed tumor tissues are particularlyvaluable. The invention can be used efficaciously even if the tumoritself displays poor immunogenicity. In addition, the invention isuseful for reducing the size of preexisting tumors, including metastaseseven after removal of the primary tumor. Finally, the invention offersthe advantage that antigen-presenting cells that are loaded with invitro transcribed RNA can be more potent vaccines than areantigen-presenting cells that are loaded with peptide antigens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating primary OVA-specific CTL induction invitro with dendritic cells pulsed with RNA. DC were pulsed with totalRNA or poly A⁺ RNA obtained from E.G7-OVA or 'EL4 cells, or in vitrotranscribed OVA RNA in the presence of the cationic lipid DOTAP asdescribed herein. DC pulsed with the OVA peptide were used forcomparison. DC and naive T cells were incubated for 5 days at a R/S of20:1. Viable lymphocytes were harvested, and the CTL activity wasdetermined in a routine europium release assay. E.G7-OVA and EL4 cellswere used as targets. This experiment was repeated three times withsimilar results.

FIG. 2 is a graph illustrating that the sensitization of E.G7-OVA RNApulsed DC for stimulation of OVA-specific primary CTL responses ismediated by the poly A⁺ fraction of RNA. DC were pulsed with total RNA,poly A⁻ RNA or poly A⁺ RNA, and cultured with naive T cells in 96-wellU-bottom plates for 5 days. The poly A⁺ RNA fraction from E.G7-OVA cellswas treated with an antisense oligonucleotide specific for the CTLepitope encoding region of the OVA gene, or a control oligonucleotidefollowed by RNase H treatment to eliminate the hybridized RNA. DC pulsedwith OVA peptide was used as a control. E.G7-OVA, EL4, and RMA cellspulsed with the OVA peptide were used as targets.

FIG. 3 is a histogram depicting the induction of anti-tumor immunity invivo in mice following a single immunization with DC pulsed with RNA. DCwere pulsed with either total or poly A⁺ RNA from E.G7-OVA cells or EL4cells, or with in vitro transcribed OVA RNA or control antisense OVARNA. Mice were immunized with 2×10⁶ DC or 5×10⁶ irradiated E.G7-OVA orEL4 cells injected intraperitoneally, followed by a challenge with 2×10⁷live E.G7-OVA cells. Mice were periodically examined for tumor growth,and were sacrificed when the tumor diameter reached 3-4 cm. All micewere sacrificed at 35-40 days post-challenge.

FIG. 4 is a histogram depicting the regression of spontaneous metastasisin mice vaccinated with DC pulsed with poly A⁺ RNA or total RNA in theB16-F10.9 melanoma model. Mice received by intrafootpad injection liveF10.9 cells, and the legs were amputated when the tumor diameter reached5.5-7.5 mm. Vaccinations were initiated 2 days post-amputation, and werefollowed by two more vaccinations at weekly intervals. Mice werevaccinated intraperitoneally with 2×10⁶ total, poly A⁻ or poly A⁺ RNApulsed DC, or irradiated F10.9 cells or F10.9/K1 cells, or PBS (as acontrol.) Mice were sacrificed based on the metastatic death in thenon-immunized or control groups (28-32 days post-amputation). Metastaticloads were assayed by weighing the lungs and by counting the number ofmetastatic nodules.

DETAILED DESCRIPTION

Before providing detailed working examples of the invention, certainparameters of the invention will be described generally.

A variety of methods are suitable for producing the tumor- orpathogen-derived RNA that can be used in the invention. As the followingexamples illustrate, it is not necessary that the RNA be provided to theAPC in a purified form. Preferably, the RNA sample (i.e., thefractionated tumor preparation or IVT RNA sample) is at least 50%, morepreferably 75%, 90%, or even 99% RNA (wt/vol). In practicing theinvention, antigen-presenting cells, preferably professional APC such asdendritic cells and macrophage, are used. Such cells can be isolatedaccording to previously-described procedures.

Any of a variety of methods can be used to produce RNA-containing tumorpreparations. For example, the tumor preparations can be produced bysonicating tumor cells in a mammalian cell culture medium such asOpti-MEM or a buffer such as phosphate buffered saline. Similarly,pathogen-derived RNA can be produced by sonicating pathogenic bacteriaor cells containing a pathogenic virus. Other methods for disruptingcells also are suitable, provided that the method does not completelydegrade the tumor- or pathogen-derived RNA. Typically, the RNApreparation has 10⁶ to 10⁸ cells/ml; most preferably 10⁷ cells/ml. Asalternatives, or in addition, to sonication, the tumor- orpathogen-derived RNA can be prepared by employing conventional RNApurification methods such as guanidinium isothiocyanate methods and/oroligo dT chromatography methods for isolating poly A⁺ RNA. IVT RNA,synthesized according to conventional methods, can be used in lieu ofRNA in tumor preparations. For example, RNA from a tumor or pathogen canbe reverse transcribed into cDNA, which then is amplified byconventional PCR techniques to provide an essentially unlimited supplyof cDNA corresponding to the tumor or pathogen RNA antigen. Conventionalin vitro transcription techniques and bacterial polymerases then areused to produce the IVT RNA. As an alternative, the IVT RNA can besynthesized from a cloned DNA sequence encoding a tumor or pathogenpolypeptide antigen. Methods for identifying such antigens are known inthe art; for example, several melanoma peptide antigens have beenidentified. RNA transcribed in vitro from cDNA encoding identifiedpeptide antigens can serve as tumor- or pathogen-specific RNA in theinvention. As an alternative, RNA can be transcribed from “minigenes”consisting of a portion of the tumor antigen cDNA that encodes anepitope. Tumor- or pathogen-specific RNA can also be produced byemploying conventional techniques for subtractive hybridization. Forexample, an RNA sample from tumor cells and non-tumor cells can be usedin the subtractive hybridization method to obtain tumor-specific RNA.

If desired, the tumor-derived or pathogen-derived RNA can be preparedfrom frozen or fixed tissues. Although not required, the tissue samplecan be enriched for tumor-specific RNA. Microdissection techniques thatare suitable for separating tumor cells from non-tumor cells have beendescribed (Zhuang et al., 1995, Cancer Res. 55:467-471; Luqmani et al.,1992, Analy. Biochem. 200:291-295; Luqmani et al., 1994, Analy. Biochem.222:102-109; Turbett et al., 1996, BioTech. 20:846-853). Once the tumorcells are separated from the non-tumor cells, tumor-derived RNA can beisolated from the tumor cells using art-known techniques. For example,tumor-derived poly-A⁺ RNA can be isolated by hybridizing the RNA to asolid phase, such as oligo(dT) linked to paramagnetic beads (see, e.g.,Raineri et al., 1991, Nucl. Acids. Res. 19:4010). The first cDNA strandthen is synthesized by reverse transcription, the RNA is removed, andthe second strand is synthesized (e.g., with DNA polymerase).Conventional in vitro transcription methods then can be used tosynthesize the RNA. Other art-known methods for amplifying RNA from asmall number of cells, or even a single cell, also can be used in theinvention.

An RNA molecule that encodes a tumor or pathogen antigenic epitope can,if desired, be engineered such that it also encodes a cell traffickingsignal sequence. Such a chimeric RNA molecule can be produced usingconventional molecular biology techniques. The chimeric RNA that isintroduced into an APC encodes a chimeric polypeptide, which contains anantigen linked to a trafficking sequence that directs the chimericpolypeptide into the MHC class II antigen presentation pathway. Forexample, the trafficking sequences employed in this embodiment of theinvention may direct trafficking of the polypeptide to the endoplasmicreticulum (ER), a lysosome, or an endosome, and include signal peptides(the amino terminal sequences that direct proteins into the ER duringtranslation), ER retention peptides such as KDEL (SEQ ID NO: 1); andlysosome-targeting peptides such as KFERQ (SEQ ID NO: 2), QREK (SEQ IDNO: 3), and other pentapeptides having Q flanked on one side by fourresidues selected from K, R, D, E, F, I, V, and L. A preferred signalpeptide that can be used in the invention is the LAMP-1 sorting signal(Wu et al., 1995, Proc. Natl. Acad. Sci. 92:11671-11675; Lin et al.,1996, Cancer Research 56: 21-26). Another example of a signal peptidethat is useful in the invention is a signal peptide substantiallyidentical to that of an MHC subunit such as class II α or β; e.g., thesignal peptide of MHC class II α is contained in the sequenceMAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO: 4). If desired, the signal peptideencoded by the RNA of the invention may include only a portion(typically at least ten amino acid residues) of the specified 25 residuesequence, provided that portion causes trafficking of the polypeptide tothe ER.

Transfection methods that are suitable for introducing the tumor- orpathogen-derived RNA into an antigen-presenting cell are known in theart. For example, 5-50 μg of RNA in 500 μl of Opti-MEM can be mixed witha cationic lipid at a concentration of 10 to 100 μg, and incubated atroom temperature for 20 to 30 minutes. Other suitable lipids includeLIPOFECTIN™ (1:1 (w/w) DOTMA:DOPE), LIPOFECTAMINE™ (3:1 (w/w)DOSPA:DOPE), DODAC:DOPE (1:1), CHOL:DOPE (1:1), DMEDA, CHOL, DDAB,DMEDA, DODAC, DOPE, DORI, DORIE, DOSPA, DOTAP, and DOTMA. The resultingRNA-lipid complex is then added to 1-3×10⁶ cells, preferably 2×10⁶,antigen-presenting cells in a total volume of approximately 2 ml (e.g.,in Opti-MEM), and incubated at 37° C. for 2 to 4 hours. Alternatively,the RNA can be introduced into the antigen presenting cells by employingconventional techniques, such as electroporation or calcium, phosphatetransfection with 1-5×10⁶ cells and 5 to 50 μg of RNA. Typically, 5-20μg of poly A⁺ RNA or 25-50 μg of total RNA is used.

When the RNA is provided as a tumor or pathogen preparation, thepreparation typically is fractionated or otherwise treated to decreasethe concentration of proteins, lipids, and/or DNA in the preparation,and enrich the preparation for RNA. For example, art-known RNApurification methods can be used to at least partially purify the RNAfrom the tumor cell or pathogen. It is also acceptable to treat the RNApreparation with proteases or RNase-free DNases. Of course, the RNA canbe synthesized using art-known nuclease-resistant analogues orderivatives in order to render the RNA less susceptible toribonucleases.

If desired, RNA encoding an immunomodulator, such as a cytokine or aco-stimulatory factor, can be introduced into the RNA-loaded APCs of theinvention. In this embodiment of the invention, the RNA encoding theimmunomodulator may be introduced into the APC prior to, simultaneouslywith, or subsequent to introduction of the tumor- or pathogen-derivedRNA. The methods described herein for introducing the tumor-derived orpathogen-derived RNA into the APC also are suitable for introducing intothe APC RNA encoding an immunomodulator (e.g., a cytokine orcostimulatory factor). Sequences encoding numerous proteins are known inthe art and can be used in the invention. If desired, RNA encoding twoor more immunomodulators can be introduced into the APC. Typically, 5-20μg of each RNA is introduced into the APC, as is described above fortumor- and pathogen-derived RNA.

The RNA-loaded antigen-presenting cells of the invention can be used tostimulate CTL proliferation in vivo or ex vivo. The ability ofRNA-loaded antigen-presenting cells to stimulate a CTL response can bemeasured or detected by measuring or detecting T-cell activation, forexample, in a conventional cytotoxicity assay. In examples providedbelow, the cytotoxicity assay entails assaying the ability of theeffector cells to lyse target cells. If desired, the target cells can beRNA-loaded APCs produced in accordance with the invention (i.e., APCsthat present an RNA-encoded cell-surface tumor or pathogen antigenicepitope that induces T cell proliferation). As is described below, thecommonly-used europium release assay can be used to assay CTLsensitization. Typically, 5-10×10⁶ target cells are labeled witheuropium diethylenetriamine pentaacetate for 20 minutes at 4° C. Afterseveral washes 10⁴ europium-labeled target cells and serial dilutions ofeffector cells at an effector:target ratio ranging from 50:1 to 6.25:1are incubated in 200 μl RPMI 1640 with 10% heat-inactivated fetal calfserum in 96-well plates. The plates are centrifuged at 500×g for 3minutes and the incubated at 37° C. in 5% CO₂ for 4 hours. A 50 μlaliquot of the supernatant is collected, and europium release ismeasured by time resolved fluorescence (Volgmann et al., J. Immunol.Methods 119:45-51, 1989).

In an alternative method for detecting CTL sensitization, an increase incytokine secretion by the CTL is detected, relative to the level ofcytokine secretion prior to contacting the CTL with an RNA-loaded APC.In situ hybridization assays, such as ELISPOT assays, can be used todetect secretion of cytokines such as TNF-α and/or γ-interferon.

EXAMPLES

The following working examples are meant to illustrate, not limit, theinvention. First, the methods used in these examples are described.

Mice

Seven to eight weeks old and retired breeder female C57BL/6 mice(H-2^(b)) were obtained from the Jackson Laboratory (Bar Harbor, Me.).

Cell Lines

The F10.9 clone of the B16 melanoma of C57BL/6 origin is a highlymetastatic, poorly immunogenic, and low class I expressing cell line.F10.9/K1 is a poorly metastatic and highly immunogenic cell line derivedby transfecting F10.9 cells with class I molecule, H-2K^(b) cDNA. RMAand RMA-S cells are derived from the Rauscher leukemia virus-induced Tcell lymphoma RBL-5 of C57BL/6 (H-2^(b)) origin. Other cell lines usedwere EL4 (C57BL/6, H-2^(b), thymoma), E.G7-OVA (EL4 cells transfectedwith the cDNA of chicken ovalbumin (OVA), A20(H-2^(d) B cell lymphoma)and L929 (H-2^(k) fibroblasts). Cells were maintained in DMEMsupplemented with 10% fetal calf serum (FCS), 25 mM Hepes, 2 mML-glutamine and 1 mM sodium pyruvate. E.G7-OVA cells were maintained inmedium supplemented with 400 μg/ml G418 (GIBCO, Grand Island, N.Y.) andF10.9/K1 cells were maintained in medium containing 800 μg/ml G418.

Antigen Presenting Cells and Responder T Cells

Splenocytes obtained from naive C57BL/6 female retired breeders weretreated with ammonium chloride Tris buffer for 3 minutes at 37° C. todeplete red blood cells. Splenocytes (3 ml) at 2×10⁷ cells/ml werelayered over a 2 ml metrizamide gradient column (Nycomed Pharma AS,Oslo, Norway; analytical grade, 14.5 g added to 100 ml PBS, pH 7.0) andcentrifuged at 600 g for 10 minutes. The dendritic cell-enrichedfraction from the interface was further enriched by adherence for 90minutes. Adherent cells (mostly dendritic cells (DC) and a fewcontaminating macrophage (Mø) were retrieved by gentle scraping, andsubjected to a second round of adherence at 37° C. for 90 minutes todeplete the contaminating Mø. Non-adherent cells were pooled as splenicDC and FACS analysis showed approximately 80%-85% DC (mAb 33D1), 1-2% Mø(mAb F4/80), 10% T cells, and <5% B Cells (data not shown).

The pellet was resuspended and enriched for Mø by two rounds ofadherence at 37° C. for 90 minutes each. More than 80% of the adherentpopulation was identified as Mø by FACS analysis, with 5% lymphocytesand <55% DC.

B cells were separated from the non-adherent population (B and T cells)by panning on anti-Ig coated plates. The separated cell population,which was comprised of >80% T lymphocytes by FACS analysis was used asresponder T cells.

Isolation of Total and Poly A⁺ Cellular RNA

Total RNA was isolated from actively growing tissue culture cells aspreviously described (Chomczynski and Sacchi, 1987, Analy. Biochem. 162:156-159). Briefly, 10⁷ cells were lysed in 1 ml of guanidiniumisothiocyanate (GT) buffer (4 M guanidinium isothiocyanate, 25 mM sodiumcitrate, pH 7.0; 0.5% sarcosyl, 20 mM EDTA, and 0.1 M2-mercaptoethanol). Samples were vortexed, and followed by sequentialaddition of 100 μl 3 M sodium acetate, 1 ml water-saturated phenol and200 μl chlorofoLm:isoamyl alcohol (49:1). Suspensions were vortexed andthen placed on ice for 15 minutes. The tubes were centrifuged at10000×g, at 4° C. for 20 minutes, and the supernatant was carefullytransferred to a fresh tube. An equal volume of isopropanol was addedand the samples were placed at −20° C. for at least 1 hour. RNA waspelleted by centrifugation as above. The pellet was resuspended in 300μl GT buffer, and then transferred to a microcentrifuge tube. RNA wasagain precipitated by adding an equal volume of isopropanol and placingthe tube at −20° C. for at least 1 hour. Tubes were microcentrifuged athigh speed at 4° C. for 20 minutes. Supernatants were decanted, and thepellets were washed once with 70% ethanol. The pellets were allowed todry at room temperature and then resuspended in TE (10 mM Tris-HCl, 1 mMEDTA, pH 7.4). Possible contaminating DNA was removed by incubating theRNA sample in 10 mM MgCl₂, 1 mM DTT and 5 U/ml RNase-free DNase(Boehringer-Mannheim) for 15 minutes at 37° C. The solution was adjustedto 10 mM Tris, 10 mM EDTA, 0.5% SDS and 1 mg/ml Pronase(Boehringer-Mannheim), followed by incubation at 37° C. for 30 minutes.Samples were extracted once with phenol-chloroform and once withchloroform; RNA was again precipitated in isopropanol at −20° C.Following centrifugation, the pellets were washed with 70% ethanol, thenair dried and resuspended in sterile water. Total RNA was quantitated bymeasuring the optical density (OD) at 260 and 280 nm. The OD 260/280ratios were typically 1.65-2.0. The RNA was stored at −70° C.

Poly A⁺ RNA was isolated either from total RNA using an OLIGOTEX™ polyA⁺ purification kit (Qiagen), or directly from tissue culture cellsusing the Messenger RNA Isolation kit (Stratagene) as per themanufacturer's protocols. If desired, alternative, conventional methodscan be used to prepare poly A⁺ RNA.

Production of In Vitro Transcribed RNA

The 1.9 kb EcoRI fragment of chicken ovalbumin cDNA in pUC18 (McReynoldset al., 1978, Nature 273:723) containing the coding region and 3′untranslated region, was cloned into the EcoRI site of pGEM4Z (Promega).Clones containing the insert in both the sense and antisenseorientations were isolated, and large scale plasmid preps were madeusing Maxi Prep Kits™ plasmid preparation kit (Qiagen). Plasmids werelinearized with BamHI for use as templates for in vitro transcription.Transcription was carried out at 37° C. for 3-4 hours using theMEGAscript In Vitro Transcription Kit™ (Ambion) according to themanufacturer's protocol and adjusting the GTP concentration to 1.5 mMand including 6 mM m⁷G(5¹)ppp(5¹)G cap analog (Ambion). Other,conventional in vitro transcription methods also are suitable. TemplateDNA was digested with RNase-free DNase 1, and RNA was recovered byphenol:chloroform and chloroform extraction, followed by isopropanolprecipitation. RNA was pelleted by microcentrifugation, and the pelletwas washed once with 70% ethanol. The pellet was air-dried andresuspended in sterile water.

RNA was incubated for 30 minutes at 30° C. in 20 mM Tris-HCl, pH 7.0, 50mM KCl, 0.7 mM MnCl₂, 0.2 mM EDTA, 100 μg/ml acetylated BSA, 10%glycerol, 1 mM ATP and 5000 U/ml yeast poly (A) polymerase (UnitedStates Biochemical). The capped, polyadenylated RNA was recovered byphenol:chloroform and chloroform extraction followed by isopropanolprecipitation. RNA was pelleted by microcentrifugation, and the pelletwas washed once with 70% ethanol. The pellet was air-dried andresuspended in sterile water. RNA was quantitated by measuring the OD at260 and 280 nm, and the RNA stored at −70° C.

Oligodeoxynucleotide Directed Cleavage of OVA mRNA by RNase H

The procedure used for RNase H site-specific cleavage of ovalbumin mRNAwas adapted from those previously described (Donis-Keller, 1979, Nucl.Acid. Res. 7: 179-192). Briefly, 5-10 μg mRNA from E.G7-OVA cells wassuspended in 20 mM HEPES-KOH, pH 8.0, 50 mM KCl, 4 mM MgCL₂, 1 mM DTT,50 μg/ml BSA and 2 μM of either the oligodeoxynucleotide 5′-CAG TTT TTCAAA GTT GAT TAT ACT-3′ (SEQ ID NO: 5), which hybridizes to sequence inOVA mRNA that codes for the CTL epitope SIINFEKL (SEQ ID NO: 6), or5′-TCA TAT TAG TTG AAA CTT TTT GAC-3′ (SEQ ID NO: 7) (Oligos, Etc.),which serves as a negative control. The samples were heated to 50° C.for 3 minutes followed by incubation at 37° C. for 30 minutes. RNase H(Boehringer-Mannheim) was added at 10 U/ml, and digestion proceeded for30 minutes at 37° C. RNA was recovered by phenol:choloroform andchloroform extraction, followed by isopropanol precipitation. RNA waspelleted by microcentrifugation, and the pellet was washed once with 70%ethanol. The pellet then was air-dried and resuspended in sterile water.Cleavage of OVA mRNA was confirmed by oligo dT primed reversetranscription of test and control samples, followed by PCR with OVAspecific primers that flank the cleavage site. PCR with actin-specificprimers was used to control between test and control samples.

Pulsing of APC

APC were washed twice in Opti-MEM medium (GIBCO, Grand Island, N.Y.).Cells were resuspended in Opti-MEM medium at 2-5×10⁶ cells/ml, and addedto 15 ml polypropylene tubes (Falcon). The cationic lipid DOTAP(Boehringer Mannheim Biochemicals, Indianapolis, Ind.) was used todeliver RNA into cells (Walker et al., 1992, Proc. Natl. Acad. Sci.U.S.A. 89: 7915-7918). RNA (in 250-500 μl Opti-MEM medium) and DOTAP (in250-500 μl Opti-MEM medium) was mixed in a 12×75 mm polystyrene tube atroom temperature (RT) for 20 minutes. The RNA to DOTAP ratio routinelyused was 1:2, and varied in certain experiments between 2:1 to 1:2. Thecomplex was added to the APC (2-5×10⁶ cells) in a total volume of 2 mland incubated at 37° C. in a water-bath with occasional agitation for 2hours. The cells were washed and used as stimulators for primary CTLinduction in vitro.

The synthetic peptide encoding the CTL epitope in chicken ovalbumin OVA,as 257-264 SIINFEKL (H-2K^(b)) (SEQ ID NO: 6), was used for peptidepulsing. The peptide had unblocked (free) amino and carboxyl ends(Research Genetics, Birmingham, Ala.). Peptides were dissolved inserum-free IMDM and stored at −20° C.

Induction of CTL In Vitro

T cells (5×10⁶ cells/ml) and RNA or peptide pulsed APC (2.5×10⁵cells/ml) were cultured in IMDM with 10% FCS, 1 mM sodium pyruvate, 100IU/ml penicillin, 100 mg/ml streptomycin, and 5×10⁻⁵ M β-mercaptoethanolin 96 well U-bottom plates to give an R/S ratio of 20:1. After 5 days,cells were used as effectors in a standard 4 hours europium releaseassay.

Cytotoxicity Assay

In these assays, 5-10×10⁶ target cells were labeled with europiumdiethylenetriamine pentaacetate for 20 minutes at 4°. After severalwashes, 10⁴ europium-labeled targets and serial dilutions of effectorcells at effector:target ratios of 50:1 to 6.25:1 were incubated in 200μl of RPMI 1640 with 10% heat-inactivated FCS in 96-well V-bottomplates. The plates were centrifuged at 500 g for 3 minutes and incubatedat 37° C. and 5% CO₂ for 4 hours. 50 μl of the supernatant washarvested, and europium release was measured by time resolvedfluorescence (Delta fluorometer, Wallace Inc., Gaithersburg, Md.).Spontaneous release was less than 25%. Standard errors (SE) of the meansof triplicate cultures was less than 5%.

Immunotherapy

E.G7-OVA Model:

C57BL/6 mice were immunized once with irradiated, RNA-pulsed APC (2×10⁶cells/mouse) or 5×10⁶ E.G7-OVA or EL4 cells. At 10-14 dayspost-immunization, mice were challenged with 2×10⁷ live E.G7-OVA cellsinjected sub-cutaneously in the flank region. Mice were monitored on aregular basis for tumor growth and size. Mice with tumor sizes >3.5 cmwere sacrificed. All survivors were sacrificed at 40 dayspost-challenge.

F10.9-B16 Melanoma Model:

Mice were received by intrafootpad injection 2×10⁵ F10.9 cells. Thepost-surgical protocol was essentially as described previously (Porgadoret al., 1995, Cancer Res. 55: 4941-4949). The legs of the mice wereamputated when the local tumor in the footpad was 5.5-7.5 mm indiameter. Post-amputation mortality was less than 5%. At two dayspost-amputation, the mice were immunized intraperitoneally, followed byweekly vaccinations twice, for a total of three vaccinations. The micewere sacrificed based on the metastatic death in the non-immunized orcontrol groups (at 28-32 days post-amputation). The metastatic loadswere assayed by weighing the lungs and by counting the number ofmetastatic nodules.

Induction of a Primary CTL Response In Vitro Using Dendritic CellsTransfected with Chicken Ovalbumin RNA.

The ability of RNA pulsed splenic dendritic cells (DC) derived fromC57BL/6 (H-2K^(b)) mice to induce a primary CTL response in vitro wasdemonstrated in the E.G7-OVA tumor system. E.G7-OVA cells were derivedfrom the EL4 tumor cell line (H-2K^(b) haplotype) by transfection withthe chicken ovalbumin cDNA (Moore et al., 1988, Cell 54: 777-785). Thechicken ovalbumin encodes a single dominant epitope (aa 257-264) inC57BL/6 mice (Rotzschke et al., 1991, Euro. Journal Immunology, 21:2891-2891).

Dendritic cells pulsed with the OVA peptide (aa 257-264) incubated withT cells from naive mice induce a potent CTL response in vitro (FIG. 1).This example demonstrates that RNA can be used as a source of antigen tosensitize DC to present antigen to CD8⁺ T cells. Splenic DC wereisolated from C57BL/6 mice and pulsed with OVA peptide or incubated withRNA synthesized in vitro (OVA IVT RNA) from a plasmid encoding thechicken ovalbumin cDNA, and used to stimulate an OVA-specific primaryCTL response in vitro. As shown in FIG. 1, both OVA peptide as well asOVA IVT RNA pulsed DC were capable of inducing an OVA specific primaryCTL response (FIG. 1). RNA pulsed DC were consistently more effectivestimulators than peptide pulsed DC. To test whether RNA isolated fromE.G7-OVA cells was capable of sensitizing DC to stimulate a primary,OVA-specific, CTL response, total RNA or poly A⁺ RNA was isolated fromE.G7-OVA or EL4 cells and incubated with DC. As shown in FIG. 1, DCpulsed with either total or poly A⁺ RNA from E.G7-OVA cells but not fromEL4 cells, were capable of inducing a strong OVA specific CTL response.Surprisingly, DC pulsed with unfractionated RNA, total or poly A⁺, wereas potent inducers of a primary CTL response as DC pulsed with the OVApeptide encoding a defined CTL epitope. Stimulation of a CTL response by(total or poly A⁺) EL4 RNA pulsed DC was only marginally abovebackground and statistically not significant (Compare to lysis of EL4targets by CTL stimulated with OVA peptide or OVA IVT RNA pulsed DC),reflecting the immunodominance of the OVA epitope and the relativeweakness of the EL4 encoded antigens.

As is illustrated by FIG. 2, total, as well as poly A⁺, but not poly A⁻,RNA isolated from E.G7-OVA cells is capable of sensitizing DC tostimulate a primary CTL response. To prove that sensitization of DC isindeed mediated by RNA, poly A⁺ RNA from E.G7-OVA cells was incubatedwith either an antisense oligonucleotide spanning the sequence encodingthe single CTL epitope present in the chicken ovalbumin gene or with acontrol oligodeoxynucleotide, and then treated with RNase H to removeany RNA sequence to which the oligodeoxynucleotide probe has hybridized.As shown in FIG. 2, induction of a primary, OVA-specific CTL responsewas abolished when the poly A⁺ RNA was incubated with the antisense, butnot with the control, oligodeoxynucleotide. FIG. 2 also shows that cellsexpressing the complete ovalbumin gene, E.G7-OVA cells, and RMA-S cellspulsed with the 8 amino acid long OVA peptide encoding the singledominant CTL epitope are lysed to a similar extent following stimulationwith total or poly A⁺ E.G7-OVA RNA pulsed DC. This indicates, therefore,that the majority of epitopes presented by E.G7-OVA RNA pulsed DCcorrespond to the previously defined single dominant CTL epitope encodedin the chicken ovalbumin gene.

Induction of Anti-Tumor Immunity by DC Pulsed with Tumor RNA.

This example demonstrates that vaccination of mice with OVA RNA pulsedDC provided protection against a challenge with E.G7-OVA tumor cells.Mice were immunized once with 2×10⁶ RNA pulsed DC or with 5×10⁶irradiated E.G7-OVA cells. Ten days later, mice were challenged with atumorigenic dose of E.G7-OVA cells. Appearance and size of the tumorwere determined on a regular basis. FIG. 3 shows the size of the tumorsat 37 days post-tumor implantation. The average tumor size in miceimmunized with irradiated EL4 cells was 25 cm, while the average tumorsize in animals immunized with the OVA expressing EL4 cells (E.G7-OVA)was only 7.03 cm. This difference is a reflection of the highimmunogenicity of the chicken OVA antigen expressed in EL4 cells and thepoor immunogenicity of the parental, EL4, tumor cell line. Vaccinationwith DC pulsed with RNA (total or poly A⁺ fraction) derived fromE.G7-OVA cells was as effective as vaccination with the highlyimmunogenic E.G7-OVA cells (average tumor size 7 cm). Vaccination withDC incubated with total or poly A⁺ RNA derived from EL4 tumor cells hada slight protective effect (average tumor size: 22 cm and 19.5 cm,respectively) which was not statistically significant, consistent withpoor to undetectable immunogenicity of EL4-derived antigens. Consistentwith the primary CTL induction data (FIG. 1), vaccination of mice withOVA IVT RNA pulsed DC provided the most effective anti-tumor response(average tumor size: 3.9 cm), while vaccination with the controlantisense OVA IVT RNA did not elicit a significant protective response.

The potency of DC pulsed with tumor-derived RNA was further evaluated inthe B16/F10.9 (H-2^(b)) melanoma metastasis model. The B16/F10.9melanoma tumor is poorly immunogenic, expresses low levels of MHC classI molecules, and is highly metastatic in both experimental andspontaneous metastasis assay systems (Porgador et al., 1996, J.Immunology 156: 1772-1780). Porgador et al. have shown that, whenvaccinations are carried out after the removal of the primary tumorimplant, only irradiated tumor cells transduced with both the IL-2 andthe H-2K^(b) genes, are capable of significantly impacting themetastatic spread of B16/F10.9 tumor cells in the lung (Porgador et al.1995, Cancer Research 55: 4941-4949). Thus, the B16/F10.9 melanoma modeland the experimental design used by Porgador et al. constitutes astringent and clinically relevant experimental system to assess theefficacy of adjuvant treatments for metastatic cancer.

To demonstrate that immunization with tumor RNA pulsed DC, in accordancewith the invention, was capable of causing the regression of preexistinglung metastases, primary tumors were induced by implantation ofB16/F10.9 tumor cells in the footpad. When the footpad reached 5.5-7.5mm in diameter, the tumors were surgically removed. Two days later, micewere immunized with irradiated B16/F10.9 cells, irradiated B16/F10.9cells transduced with the H-2K^(b) gene (F10.9K1), or with RNA pulsed DCpreparations (FIG. 4). The mice received a total of three vaccinationsgiven at weekly intervals. The average lung weight of a normal mouse is0.18-0.22 g. Mice treated with PBS (a negative control) were overwhelmedwith metastases. The mean lung weight of mice in this treatment groupwas 0.81 g; approximately three-quarters of the weight was contributedby the metastases, which were too many to count (>100 nodules). Asimilar metastatic load was seen when mice were treated with irradiatedB16/10.9 cells (data not shown), which confirms numerous previousobservations that treatment with irradiated B16/F10.9 tumor cells alonehas no therapeutic benefit in this tumor model. As also previouslyshown, immunization with H-2K^(b) expressing B16/F10.9 cells (F10.9K1,as a positive control) had a modest therapeutic benefit, as indicated bya statistically significant decrease in the average lung weight of theanimals in this treatment group. A dramatic response, however, was seenin animals treated with DC that were pulsed with total RNA derived fromF10.9 cells in accordance with the invention. The mean lung weight ofmice in this treatment group was 0.37 g. A significant dramatic responsealso was seen in mice treated with DC pulsed with poly A⁺ RNA derivedfrom F10.9 cells in accordance with the invention (average lung weight:0.42 g). By contrast, no statistically significant decrease inmetastatic load was seen in mice treated with DC that were pulsed witheither the poly A⁻ RNA fraction derived from F10.9 cells or with totalRNA isolated from EL4 tumor cells.

The observation that cells expressing the OVA protein (E.G7-OVA) orcells pulsed with the OVA peptide were efficiently lysed by CTL, and thesensitization of DC fractionated with poly A⁺ RNA, strongly suggest thatRNA-mediated stimulation of CTL occurs via translation of the input RNAand generation of the predicted class I restricted epitopes, in thiscase a single dominant epitope encoded in the chicken OVA peptide. Thesedata show that RNA mediated sensitization of DC is more effective thanpulsing with peptide because the transfected RNA can serve as acontinuous source for the production of antigenic peptides.

Therapeutic Use

The invention can be used to treat or prevent tumor formation in apatient (e.g., melanoma tumors, bladder tumors, breast cancer tumors,colon cancer tumors, prostate cancer tumors, and ovarian cancer tumors).Similarly, the invention can be used to treat or prevent infection in apatient with a pathogen such as a bacterium (e.g., Salmonella, Shigella,or Enterobacter) or a virus (e.g., a human immunodeficiency virus, aHerpes virus, an influenza virus, a poliomyelitis virus, a measlesvirus, a mumps virus, or a rubella virus).

In treating or preventing tumor formation or pathogen infection in apatient, it is not required that the cell(s) that is administered to thepatient be derived from that patient. Thus, the antigen-presenting cellcan be obtained from a matched donor, or from a culture of cells grownin vitro. Methods for matching haplotypes are known in the art.Similarly, it is not required that the RNA be derived from the patientto be treated. RNA from a donor can be used.

It is preferable that treatment begin before or at the onset of tumorformation or infection, and continue until the cancer or infection isameliorated. However, as the examples described herein illustrate, theinvention is suitable for use even after a tumor has formed, as theinvention can cause a regression of the tumor. In treating a patientwith a cell or vaccine produced according to the invention, the optimaldosage of the vaccine or cells depends on factors such as the weight ofthe mammal, the severity of the cancer or infection, and the strength ofthe CTL epitope. Generally, a dosage of 10⁵ to 10⁸ RNA-loadedantigen-presenting cells/kg body weight, preferably 10⁶ to 10⁷ cells/kgbody weight, should be administered in a pharmaceutically acceptableexcipient to the patient. The cells can be administered by usinginfusion techniques that are commonly used in cancer therapy (see, e.g.,Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimaldosage and treatment regime for a particular patient can readily bedetermined by one skilled in the art of medicine by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

Where the antigen-presenting cell is used to induce a CTL response invitro, the resulting effector CTLs can subsequently be administered to amammal in a CTL-based method of therapy (see, e.g., PCT/US91/06441). CTLproduced in vitro with the antigen-presenting cells of the invention canbe administered in a pharmaceutically acceptable excipient to a mammalby employing conventional infusion methods (see, e.g., Rosenberg et al.,supra). Typically, 10⁹-10¹⁰ cells are administered over the course of 30minutes, with treatment repeated as necessary. Such a CTL-based methodof therapy may be combined with other methods, such as directadministration of the antigen-presenting cells of the invention. The CTLand antigen-presenting cells may be autologous or heterologous to thepatient undergoing therapy. If desired, the treatment may also includeadministration of mitogens (e.g., phyto-hemagglutinin) or lymphokines(e.g., IL-2 or IL-4) to enhance CTL proliferation.

What is claimed is:
 1. An isolated RNA-pulsed antigen presenting cellcomprising pathogen specific RNA, wherein the pathogen specific RNAcomprises in vitro transcribed RNA.
 2. The antigen presenting cell ofclaim 1, wherein said antigen presenting cell is a dendritic cell. 3.The antigen presenting cell of claim 1, wherein the pathogen isSalmonella, Shigella, human immunodeficiency virus, herpes virus,influenza virus, poliomyelitis virus, measles virus, mumps virus orrubella virus.
 4. The antigen presenting cell of claim 1, wherein theantigen presenting cell and the pathogen RNA are derived from the samepatient.
 5. The antigen presenting cell of claim 1, wherein at least 80%of the pathogen specific RNA is polyA⁺.
 6. The antigen presenting cellof claim 5 wherein at least 90% of the pathogen specific RNA is polyA⁺.7. The antigen presenting cell of claim 1, wherein the pathogen specificRNA is pathogen-derived RNA.
 8. The antigen presenting cell of claim 1,wherein said in vitro transcribed RNA is capped.
 9. The antigenpresenting cell of claim 1, wherein said in vitro transcribed RNA ispolyadenylated.
 10. The antigen presenting cell of claim 9, wherein theRNA is in vitro transcribed from a cDNA template made by reversetranscriptase of pathogen RNA.
 11. The antigen presenting cell of claim10, wherein said cDNA template is amplified by PCR.
 12. The antigenpresenting cell of claim 1, further comprising RNA encoding animmunomodulator.
 13. The antigen presenting cell of claim 12, whereinthe immunomodulator is a cytokine.
 14. The antigen presenting cell ofclaim 12, wherein said immunomodulator is a costimulatory factor. 15.The antigen presenting cell of claim 1, wherein said pathogen specificRNA also encodes a cell trafficking signal sequence.
 16. The antigenpresenting cell of claim 15, wherein the cell trafficking signal directsan antigenic epitope encoded in the RNA to the endoplasmic reticulum, alysosome, or an endosome within the cell.
 17. The antigen presentingcell of claim 15, wherein the cell trafficking signal is a LAMP signal.18. The antigen presenting cell of claim 3, wherein the pathogen ishuman immunodeficiency virus.
 19. A therapeutic composition comprisingthe antigen presenting cell of claim 1.